Seroprevalence and risk factors for Neospora spp. infection in equine in Egypt

Neospora infections in equine are associated with reproductive disorders and neurological diseases. Nevertheless, Egypt has no epidemiological information on this parasite in equine. This study determined the prevalence of Neospora spp. infection in 325 equines from three Egyptian governorates located at Northern Egypt using cELISA. The prevalence of antibodies against Neospora spp. was 19% (95% CI: 14.09–25.05) in horse, 34.1% (95%CI: 24.92–44.69) in donkey and 26.7% (95% CI: 15.97–41.04) in mule. In comparison to horse, donkey had a considerably higher chance of contracting Neospora spp. infection (OR = 1.80, 95% CI: 0.78–4.13; P = 0.016). The risk was also higher in freely grazing animals (OR = 3.49, 95% CI: 0.95–12.78; P = 0.059). Moreover, yearling animals (12–24 months) (OR = 5.03, 95% CI: 1.51–16.80; P = 0.009) and those with natural breeding (OR = 11.80, 95% CI: 3.24–42.99, P < 0.0001) and a history of early abortion (OR = 7.04, 95% CI: 3.01–16.47; P < 0.0001) also showed a greater risk of seropositivity. The risk of Neospora infection increased significantly in equines contacted with dogs (OR = 5.16, 95% CI: 1.76–15.10; P = 0.003). This the first serological study to determine the seroprevalence of Neospora spp. in equine in Egypt. Further studies are necessary to identify the species of Neospora and to understand the role of above-mentioned risk factors in spreading of infection.

www.nature.com/scientificreports/In a serological study on horses in Israel, the seroprevalence of Neospora spp. was 11.9% in apparent healthy horses but it was significantly higher among horses had neurological symptoms (21.2%) or in mares suffered from abortion (37.5%) 5 .In addition, other studies were conducted in Brazil to investigate antibodies against Neospora spp., Whereas Villalobos et al. 6 found a prevalence of 15.1% in animals raising in So Paulo state but was 2.5% in serum samples from ten federal states as reported by Hoane et al. 14 .
Animals infected with N. caninum have been identified using a variety of laboratory procedures.In order to identify specific antibodies in the sera of infected animals, a range of serology procedures are employed, including the indirect fluorescent antibody test (IFAT), direct agglutination tests, immunoblotting (IB), and ELISAs.Moreover, N. hughesi can be differentiated from N. caninum by DNA biotechnology [16][17][18][19] .We used ELISA to establish the long-term stability of high antibody levels against the parasite in equine 20 .Serologic testing has a lower cost advantage than other tests.ELISA is the most suited serologic technique for high throughput screening of antibodies to this parasite 21 .
In Egypt, the prevalence and associated risk factors for Neospora infection have been studied in domestic ruminants.The prevalence rates were ranged between 8.6 and 36.1% in sheep 22,23 , 10.9% in camels 24 and 20.43-24.6% in dairy cattle 25,26 .However, there is a lack of epidemiological information about the disease in equine.
Therefore, the goal of the study was to determine the seroprevalence of Neospora spp. in equine and to identify the associated risk factors for Neospora infection in equine in three Egyptian governorates.

Study area
The study was conducted on equine raising in three governorates namely Giza, Kafr ElSheikh and Qalyubia which situated at North of Egypt.The selected governorates are distinguished by having a high equine population where equine is used in tourism around the pyramid area in the Giza governorate.In addition, equine is mostly utilized in breeding and droughting in other governorates, including Kafr ElSheikh and Qalyubia.
Giza governorate situated at west of Nile and southwest to Cairo.Giza's climate is hot and dry (Köppen: BWh); the average annual temperature is 20 °C, but the summer months can reach a maximum of 40 °C.There is also rare precipitation throughout the year.On the other side, the governorates of Kafr ElSheikh and Qalyubia located at Egypt's Nile Delta, which are distinguished by a year-round average temperature of 28 °C and by cold temperatures and minimal precipitation in the winter.

Study design
Across sectional study was performed during January to December 2022.Blood samples from 325 asymptomatic equine were taken by convenience from three governorates in Egypt: 195 horses, 85 donkey, and 45 mules.These animals were asymptomatic and selected randomly from the three governorates under the study.
Blood sample (5 mL) were taken from jugular vein of each animal using vacutainer, which transferred in icebox to veterinary Diagnostic Laboratory, Faculty of Veterinary Medicine, Benha University.Sera were separated by centrifugation at 3000 × g for 10 min, and then stored at − 20 °C until serological examination.During sampling, data of each examined animal were taken including equine type (horse, mule and donkey), age (12 months, 12-24 months, and > 24 months old), sex (male and female) and location, Table 1.A questionnaire was prepared to collected data of other management factors like type of animal management or breeding, history of abortion and contact with dogs.

Serological analysis
All sera were examined for the existence of antibodies against Neospora spp.using a commercial competitive inhibition enzyme-linked immunosorbent assay, (cELISA, VMRD, Inc., Pullman, WA, USA).Serological testing and evaluation were carried out in accordance with the manufacturer's recommendations.The optical densities (ODs) were measured using ELISA plate reader AMR-100 (AllSheng, Chnia) at 650 nm.This test has a sensitivity and specificity of 91.4% and 99.4%, respectively 27 .Moreover, the manufacturer provided negative and positive control with cELISA kit.After calculating the percent inhibition, samples had an inhibition of less than 30% considered positive.

Statistical analysis
Data were analyzed using the statistical SPSS software version 24 (IBM, SPSS Inc., Chicago, IL, USA).Data on seroprevalence of Neospora spp.were statistically evaluated, taking into consideration factors like location, gender, age, species, type of breeding, type of management, history of abortion, and contact with dogs.Using the Chi-square test, the association between neosporosis and other risk variables was evaluated.The results considered significant in case of P value less than 0.05.The multivariate logistic regression model was used to assess the independent risk factors of each explanatory variable for all variables that had a P value in the univariate analysis of less than 0.25 28 .The occurrence odds ratio (OR) and associated 95% confidence interval (CI) were calculated by multivariate logistic regression [29][30][31][32] .The goodness-of-fit of the model was evaluated using the Hosmer-Lemeshow statistic 33 .

Ethics approval and consent to participate
The study protocol followed the guidelines of the ethical committee of the Faculty of Veterinary Medicine at Benha University, which complies with all Egyptian regulations regarding research and publication.Serum samples were collected and handled in accordance with the Committee's Animal Ethical Standards and Guidelines.The procedures of present study were conducted in accordance with the ARRIVE recommendations.

Results
Among the 325 equine samples, 78 (or 24%) tested positive for IgG antibodies to Neospora spp.The prevalence rate was statistically differed (P < 0.05) between various equine hosts, it was 19% in horse, 34.1% in donkey, and 26.7% for mule.However, no-statistical difference (P > 0.05) was observed between sex.Moreover, there were statistically different in seropositivity for Neospora spp. between age groups (P < 0.05).Antibodies against Neospora spp.were found in 7.7% of foals, 31.2% of yearlings, and 18.4% of adults, Table 2.
The management of equine showed significant impact (P < 0.05) on seroprevalence of Neospora spp., where only 8% of equine maintained in stables tested positive for antibodies to Neospora spp., compared to 34% of equine that were allowed to graze in pasture.
We evaluated the effect of breeding to understand its role in prevalence of antibodies to Neospora spp.We found that the seroprevalence was 4.6% in equine that had artificial insemination and 34% in those had natural breeding.The seroprevalence was significantly influenced by the type of breeding, Table 2.
For animals with and without a history of abortion, there was a substantial difference in seropositivity (P < 0.0001) between the two groups.The history of abortion in 36 female animals was recorded.The seropositivity rate was found higher in equine (67.6%) with early abortion history than animals with no abortion history (12.9%),Table 2.
The multivariable logistic regression analysis was tabulated in Table 3.The results revealed that the risk of Neospora spp.infection in mule and donkey was two times higher than in horse.Yearling equine of 12-24 months age was five times (OR = 5.03, 95% CI: 1.51-16.80)more likely to be infected than foals.Moreover, grazing horses with history of early abortion were three and seven times more likely to be infected when compared with equine kept in stable or equine with no history of abortion.Additionally, natural breeding and contact with dogs in examined equine increased risk of Neospora spp.infection eleven and five times more compared to AI and absence of dogs.

Discussion
Neosporosis is one of the parasite diseases that affects domestic animals and poses a risk to the public's health in addition to inflicting severe economic losses 34,35 .Neospora spp.infections either subclinical or clinical forms have been reported horses from numerous countries across the world [36][37][38] .In Egypt, many animal species, including cattle, sheep, goats, and camels, have been found to be infected with Neospora 23,24 .However, to our knowledge, no earlier research has identified infections with Neospora spp. in equine in Egypt.Therefore, the present study aims to investigate the presence of antibodies against Neospora spp. in equine in three Egyptian governorates to improve the epidemiology of the disease in these areas and to determine the risk factors that infection poses.
The overall findings of the current study show that 24% of equines in Egypt had antibodies against Neospora spp., which are consistent with the stated rate (24%) in horses from the Czech Republic 39 .
In comparison to the prevalence rates of diseases among horse in earlier studies, the seropositive rate of Neospora infection in horse (19%) detected in the current study is relatively high by comparison with other studies from Brazil.The prevalence rate was 2.5% in examined horse from ten different Brazilian states according to  40 .
On the other hand, reports from other countries indicate higher prevalence rates.A prevalence of 34% was reported among Chilean horses by Patitucci et al. 41 .For horse of various ages in Italy, Ciaramella et al. 42 reported a prevalence of 28%.In contrast, animals from Costa Rica were found to have a prevalence rate of 3.5% 43 .
Additionally, when indirect fluorescent antibody test (IFAT) was used, it was discovered that horses from various Italian regions had a prevalence of 28% 42 and 10% 44 , whereas horses from Israel had a prevalence of 12% 5 .In France, a direct agglutination test on horses yielded a frequency of 23% 45 .
Furthermore, the antibodies against Neospora spp.were detected in 34.1% of donkey and 26.7% of mule using cELISA, which is consistent with the percentages seen in donkeys and mules from southern Punjab, Pakistan 27 , it was 32.5% and 26.9%, respectively.This high seropositivity when compared to the 2% and 0.4% prevalence rate of Neospora spp.found in donkeys from the northeastern part of Brazil 46,47 .
According to previous literatures, it is clear that the prevalence of Neospora spp. in equine varied significantly between and even within countries.These variances may result from the use of different serological tests in each study, the testing methodologies' limitations, non-standard controls, and the applied cut-off points 2,17,23,[52][53][54][55][56] .Moreover, Neospora spp.seroprevalences may be influenced by the study design, sample collection criteria, varying amounts of exposure to various risks, and protective factors for infection or sickness, which makes it challenging to compare the findings from diverse studies 36,57,58 .
Neospora spp.seroprevalence varied significantly between age groups and the highest seroprevalence was observed among equine of 12-24 months age, according to the current study.This finding comes in agreement with the earlier study from Israel 5 , which reported a significant greater seroprevalence to Neospora spp. in older horses due to an increased arisk of exposure via horizontal spreading over time.In addition, Neospora spp.seroprevalence increased non-significantly (22.2% and 27.2%) between the age groups of 1-10 and 11-20 years old, respectively, in a Turkish study 59 .Contrary to our findings, Bártová et al. 60 observed that there was no significant difference between equine age groups and the prevalence rose with equine age, ranging from 15.4% in foals to 26.6% in yearlings, indicating postnatal transmission.
In the line of previous results of Villalobos et al. 6 and Talafha et al. 61 , there was no significant difference (P > 0.05) between both sexs.The current findings revealed higher prevalence in females which attributed to females may be more susceptible to Neospora spp.infection due to a worse immune response during different times of their life as aresult of stress of pregnancy and lacation, which causes an immunosuppression and increases their vulnerability to infection 62,63 .
Concerning to feeding type of examined equids, the present results were consistent with previous findings of Nazir et al. 27 , where the Neospora spp.seroprevalence increased significantly among grazing animals.This could be explained by horizontal transmission of Neospora spp.among equids because it is considered as main rout of transmission and increase risk of infection in equids 5 .
The current findings revealed that the natural breeding increased the risk of infection in equine when compared to AI, which was consistent was findings of de Araújo Valença et al. 64 .This explain the vertical transimission play an vital role in transmission of Neospora spp. in equine 4 .
In comparison to female equines without a history of abortion, the prevalence of Neospora spp. was significantly (P < 0.05) greater in females with history of abortion.However the evidence is rather scant compared to cattle, where N. caninum has been consistently shown to be a major cause of abortion, Neospora species may play a role in equine abortions 6 .Our results are consistent with other researches in which a population of mares with reproductive problems had high percentage of anti-Neospora spp.antibodies 45 .
The results demonstrated that the equines contacted with dogs had high signifcant prevalence rate for Neospora spp.when comapred with other animals.These results are consistent with those that reported by de Araújo Valença et al. 64 .The existence of seropositive dogs around the investigated equids may indicate that the animals were exposed to the parasite, increasing the risk of oocyst contamination of food or water and subsequent horizontal transmission 8,43 .
The current study's limitations include the bias in sample collection for possible representation of the examined governorates or equine population and the diagnostic approach since it can identify the Neospora spp.However, we were unable to determine the species of Neospora.As a result, more research is required for largescale screening and determining Neospora species in Egyptian equines.

Conclusion
This is the first instance of Neospora spp.being found in Egyptian equines.Several risk factors are associated with Neospora spp.infections like age, species, management, type of breeding, history of abortion and contact with dogs.The findings demonstrate that Neospora spp. is a contributing factor in abortions in Egyptian mares.Thus, the prevention and control program should be implemented to prevent the spreading of the diseases among equine and economic losses as a result of abortion.Additional epidemiological researches for molecular detection of parasite DNA in tissue from placentas or aborted fetus to identify Neospora spp.are necessary.

Table 1 .
Descriptive analysis for factors used to determine the seroprevalence of Neospora infection in equine.

Table 2 .
Univariable analysis for factors used to determine the seroprevalence of Neospora infection on equine.
*The result is significant at P < 0.05.Variable Total